Caspase 3 is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes that undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. This protein cleaves and activates caspases 6 and 7; and the protein itself is processed and activated by caspases 8, 9, and 10. Caspase 3 can hydrolyze the peptide substrate acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA) resulting in the release of the p-nitroaniline (pNA) moiety. p-Nitroaniline has a high absorbance at 405nm. Thus the activity of recombinant human caspase 3 can be measured by calculate the concentration of the pNA released from the substrate. The reaction was performed in adding 50μL 2×buffer (50mM HEPES, 100mM NaCl, 10mM DTT, 2mM EDTA, 10% glycerol) to 96 well plates, then add 50μL various concentration of caspe 3 (diluted by 1×buffer, 25mM HEPES, 50mM NaCl, 5mM DTT, 1mM EDTA, 5% glycerol) to each well, finally, add 5μL 4mmol Ac-DEVD-pNA to each well. Cover the 96 well plates and incubate at 37℃ for 2h. p-Nitroaniline (pNA) standard curve prepare by double dilute 200μM pNA with 1×buffer and record the OD value at 405nm. Calculate the caspase 3 activity in pmol of pNA released per min per μg recombinat human caspase 3.
The specific activity of recombinant human caspase 3 is 2196pmol/min/μg.